During the second half of the XIX century, several species of red wood ants ( Formica rufa group) were used as biological control agents and introduced to the Italian Apennine mountains where they were formerly absent. Among these, Formica lugubris was the most widely introduced alien species. In 1996, however, a taxonomic revision defined two sibling species, F. lugubris and F. paralugubris , each with distinct ecological and behavioural characteristics that differently affect their spatial distribution. The first one can be both monodomous and polydomous with a low number of interlinked nests, and it is obliged to mate by nuptial flight. Conversely, F. paralugubris is usually unicolonial, with a considerable portion of intranest mates. Discrimination between the two species using morphological characters is possible but requires considerable taxonomic expertise. Restriction analysis, whereby presence/absence of a BamHI site in the mitochondrial DNA COI gene results in different restriction profiles, provides an alternative efficient method to unambiguously distinguish F. lugubris from F. paralugubris . We applied this method to identify ant species introduced to several locations in the Campigna Forest of the Foreste Casentinesi, Monte Falterona and Campigna National Park, one of the Italian locations mostly affected by alien ant introductions. This area has considerable ecological relevance. Given the potential impact of ants on native arthropod communities, it is of the most importance to identify which species has been introduced. Contrary to previous assumptions, results of our analysis clearly show that only F. paralugubris is present in the National park.